Change in intestinal alkaline phosphatase activity is a hallmark of antibiotic-induced intestinal dysbiosis.

OBJECTIVE: Intestinal alkaline phosphatase (IAP) maintains intestinal homeostasis by detoxifying bacterial endotoxins and regulating gut microbiota, and lipid absorption. Antibiotics administered to animals can cause gut dysbiosis and barrier disruption affecting animal health. Therefore, the present study sought to investigate the role of IAP in the intestinal environment in dysbiosis. METHODS: Young male mice aged 9 weeks were administered a high dose of antibiotics to induce dysbiosis. They were then sacrificed after 4 weeks to collect the serum and intestinal organs. The IAP activity in the ileum and the level of cytokines in the serum samples were measured. Quantitative real-time polymerase chain reaction analysis of RNA from the intestinal samples was performed using primers for tight junction proteins (TJPs) and proinflammatory cytokines. The relative intensity of IAP and toll-like receptor 4 (TLR4) in intestinal samples was evaluated by western blotting. RESULTS: The IAP activity was significantly lower in the ileum samples of the dysbiosisinduced group compared to the control. The interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha concentrations were significantly higher in the ileum samples of the dysbiosis-induced group. The RNA expression levels of TJP2, claudin-3, and claudin-11 showed significantly lower values in the intestinal samples from the dysbiosis-induced mice. Results from western blotting revealed that the intensity of IAP expression was significantly lower in the ileum samples of the dysbiosis-induced group, while the intensity of TLR4 expression was significantly higher compared to that of the control group without dysbiosis. CONCLUSION: The IAP activity and relative mRNA expression of the TJPs decreased, while the levels of proinflammatory cytokines increased, which can affect intestinal integrity and the function of the intestinal epithelial cells. This suggests that IAP is involved in mediating the intestinal environment in dysbiosis induced by antibiotics and is an enzyme that can potentially be used to maintain the intestinal environment in animal health care.

The bisphenol S contamination level observed in human follicular fluid affects the development of porcine oocytes.

Bisphenol S (BPS), the main replacement for bisphenol A (BPA), is thought to be toxic, but limited information is available on the effects of Bisphenol S on ovarian follicles. In our study, we demonstrated the presence of Bisphenol S in the follicular fluid of women at a concentration of 22.4 nM. The effect of such concentrations of Bisphenol S on oocyte maturation and subsequent embryo development is still unknown. Therefore, we focused on the effect of Bisphenol S on in vitro oocyte maturation, fertilization, and embryo development. As a model, we used porcine oocytes, which show many physiological similarities to human oocytes. Oocytes were exposed to Bisphenol S concentrations similar to those detected in female patients in the ART clinic. We found a decreased ability of oocytes to successfully complete meiotic maturation. Mature oocytes showed an increased frequency of meiotic spindle abnormalities and chromosome misalignment. Alarming associations of oocyte Bisphenol S exposure with the occurrence of aneuploidy and changes in the distribution of mitochondria and mitochondrial proteins were demonstrated for the first time. However, the number and quality of blastocysts derived from oocytes that successfully completed meiotic maturation under the influence of Bisphenol S was not affected.

Inactivation of mammalian spermatozoa on the exposure of TiO2 nanorods deposited with noble metals.

Titanium dioxide (TiO2) nanorods (NRs) are well-known semiconducting and catalytic material that has been widely applied, but their toxicities have also attracted recent interest. In this study, we investigated and compared the toxic effects of TiO2 NRs and TiO2 NRs loaded with Ag or Au NPs on boar spermatozoa. As a result, sperm incubated with Ag-TiO2 NRs showed lower motility than sperm incubated with controls (with or without TiO2 NRs) or Au-TiO2 NRs. In addition, sperm viability and acrosomal integrity were defective in the presence of Ag-TiO2 NRs, and the generation of intracellular reactive oxygen species (ROS) increased significantly when spermatozoa were incubated with 20 µg/ml Ag-TiO2 NRs. We discussed in depth the charge transfer mechanism between enzymatic NADPH and Ag-TiO2 NRs in the context of ROS generation in spermatozoa. The effects we observed reflected the fertilization competence of sperm incubated with Ag-TiO2 NRs; specifically sperm penetration and embryonic development rates by in vitro fertilization were reduced by Ag-TiO2 NRs. To summarize, our findings indicate that exposure to Ag-TiO2 NRs could affect male fertilization fecundity and caution that care be exercised when using these NRs.

Combinatorial Effect of Dietary Oregano Extracts and 3,4,5-Trihydroxy Benzoic Acid on Growth Performance and Elimination of Coccidiosis in Broiler Chickens.

We aimed to compare the combinatorial effect of 3,4,5-trihydroxybenzoic acid (THB) and oregano extracts (OE) with THB alone on the growth performance and elimination of deleterious effects in coccidiosis-infected broilers. A total of 210 one-day-old broilers were randomly assigned to one of five dietary treatments, with six replicates each, for 35 days. Dietary treatments were: 1) non-challenged, non-treated (NC); 2) challenged, non-treated (PC); 3) PC+ Salinomycin (0.05 g/kg; AB); 4) PC+THB (0.1 g/kg; THB); and 5) PC+THB+OE (0.1 g/kg; COM). On day 14, all groups except for NC were challenged with a 10-fold dose of Livacox® T anticoccidial vaccine to induce mild coccidiosis. All treatments significantly improved (P<0.05) body weight, average daily gain, and average daily feed intake, compared to PC, on days 21, 28, and 35. However, all treatments significantly reduced (P<0.05) the feed conversion ratio of PC by more than 14.60% on day 35, 11.76% during growing period, and 10.36% through the entire period. Broilers receiving anticoccidial treatments had 54.23% and 51.86% lower lesion scores (P<0.05) at 4 and 7 days post-infection, respectively, compared to PC. Additionally, the villus height of COM was significantly longer (P < 0.05) than that of THB. Although the molecular action of COM remains unclear, OE addition to THB reduced the shedding of oocysts better than THB alone (P<0.05, 9-11 days post-infection). Most importantly, COM effectively minimized the mortality of challenged birds from as high as 11.90% (PC) to 0%, a level similar to NC and AB, while THB maintained a mortality of 2.38%. In conclusion, the anticoccidial effect of THB can be enhanced by the addition of OE for better animal performance and the elimination of deleterious effects from coccidiosis-infected broilers for 35 days.

Broiler responses to dietary 3,4,5-trihydroxybenzoic acid and oregano extracts under Eimeria challenge conditions.

This study was conducted to evaluate the efficacy of a combination 3,4,5-trihydroxybenzoic acid (THB) and oregano extracts (i.e., Carvacrol and Thymol) at intake/dietary different levels on growth performance, intestinal health indicators, immune responses and fecal oocyst shedding in broiler chickens under Eimeria challenged condition. A total of 336 one-day-old broilers were randomly assigned to one of six dietary treatments with seven replications per treatment. Dietary treatments were: i) Non-challenged bird without any dietary treatment (NCNT), ii) Challenged bird without any dietary treatment (CNT), iii) Challenged birds fed a THB diet (0.1 g/kg, THB), iv) Challenged birds fed a combination of THB and oregano extracts diet (0.1 g/kg, COM 100), and a gradual increase of combination of THB and oregano extracts likely v) 0.15 g/kg (COM 150), and 0.2 g/kg (COM 200). On day 14, all groups except for NCNT have orally challenged with a 10-fold dose of Livacox® T anticoccidial vaccine to trigger coccidiosis. The results indicated that Eimeria-challenged broilers fed COM 100 and COM 200 diets increased (p < 0.05) body weight than CNT diet on day 35. Furthermore, birds fed COM 100 and COM 200 diets increased (p < 0.05) average daily gain compared to those fed CNT diets for the entire experimental period. There is no significant (p > 0.05) in average daily feed intake, feed efficiency between NCNT and birds fed with combined THB and oregano extracts for the entire experimental period. A combination of THB and oregano extract regardless of concentration levels or THB alone reduced (p < 0.05) lesion score in ileum compared to the CNT diet for 7 days post-infection (dpi). Birds fed COM 100 diet had lower (p < 0.05) intestinal lesion scores in jejunum and caeca on 7 dpi compared to those were in the CNT diet. No (p > 0.05) difference was observed in the oocysts per gram of feces count, intestinal morphology, carcass traits and blood cytokine concentration among the infected treatments. Collectively, we conclude that birds fed with a combination of THB and oregano extracts regardless of the ratios that were used demonstrated better recovery of health after the coccidial challenge than using only THB alone.

Mammalian Cell-Free System Recapitulates the Early Events of Post-Fertilization Sperm Mitophagy.

Propagation of paternal sperm-contributed mitochondrial genes, resulting in heteroplasmy, is seldom observed in mammals due to post-fertilization degradation of sperm mitochondria, referred to as sperm mitophagy. Whole organelle sperm mitochondrion degradation is thought to be mediated by the interplay between the ubiquitin-proteasome system (UPS) and the autophagic pathway (Song et al., Proc. Natl. Acad. Sci. USA, 2016). Both porcine and primate post-fertilization sperm mitophagy rely on the ubiquitin-binding autophagy receptor, sequestosome 1 (SQSTM1), and the proteasome-interacting ubiquitinated protein dislocase, valosin-containing protein (VCP). Consequently, we anticipated that sperm mitophagy could be reconstituted in a cell-free system consisting of permeabilized mammalian spermatozoa co-incubated with porcine oocyte extracts. We found that SQSTM1 was detected in the midpiece/mitochondrial sheath of the sperm tail after, but not before, co-incubation with oocyte extracts. VCP was prominent in the sperm mitochondrial sheath both before and after the extract co-incubation and was also detected in the acrosome and postacrosomal sheath and the subacrosomal layer of the spermatozoa co-incubated with extraction buffer as control. Such patterns are consistent with our previous observation of SQSTM1 and VCP associating with sperm mitochondria inside the porcine zygote. In addition, it was observed that sperm head expansion mimicked the early stages of paternal pronucleus development in a zygote during prolonged sperm-oocyte extract co-incubation. Treatment with anti-SQSTM1 antibody during extract co-incubation prevented ooplasmic SQSTM1 binding to sperm mitochondria. Even in an interspecific cellular environment encompassing bull spermatozoa and porcine oocyte extract, ooplasmic SQSTM1 was recruited to heterospecific sperm mitochondria. Complementary with the binding of SQSTM1 and VCP to sperm mitochondria, two sperm-borne pro-mitophagy proteins, parkin co-regulated gene product (PACRG) and spermatogenesis associated 18 (SPATA18), underwent localization changes after extract coincubation, which were consistent with their degradation observed inside fertilized porcine oocytes. These results demonstrate that the early developmental events of post-fertilization sperm mitophagy observed in porcine zygote can be reconstituted in a cell-free system, which could become a useful tool for identifying additional molecules that regulate mitochondrial inheritance in mammals.

Artemisia vulgaris extract causes precocious acrosome reaction and viability loss but low rate of membrane damage in mouse spermatozoa.

Several herbs including Artemisia are known to possess conceptive property. In the present study, mouse spermatozoa were incubated with ethanol extract of Artemisia vulgaris leaves. The effect of extract on acrosome exocytosis was studied by labeling spermatozoa with fluorescein isothiocyanate (FITC) peanut agglutinin and by staining with Coomassie blue. Viability and membrane integrity were studied by Trypan-blue staining and hypo-osmotic swelling test. Artemisia extract at very low concentration caused precocious acrosome reaction and loss of sperm viability. Acrosome reaction increased remarkably from 22.63% to 88.42% with increasing extract concentration from 0 to 2,000 µg/mL. However, the viability loss of spermatozoa was increased from 11.71% in control to 63.73% in samples treated, evaluated by Trypan-blue staining method. Membrane damage caused by the extract, evaluated by hypo-osmotic swelling test was even low, ranging from 2.27% to only 24.23%. These results indicate that Artemisia extract might block fertilization by causing precocious acrosome exocytosis in spermatozoa. A direct contraceptive effect was tested by injecting the plant extract into the vagina of female mice and then allowing them to mate with normal males. The treated female mice delivered significantly fewer litters in comparison to the control.

Effect of calcium stearoyl-2 lactylate and lipase supplementation on growth performance, gut health, and nutrient digestibility of broiler chickens.

OBJECTIVE: To evaluate calcium stearoyl-2 lactylate (CSL) performance as an exogenous emulsifier together with lipase for broiler diets. METHODS: In total, 252 one-day-old Ross 308 broiler chickens were allocated in a completely randomized design to give 6 replications per treatment with 7 birds in each cage. There were six dietary treatments representing a 2×3 factorial arrangement consisted of two energy levels (standard energy [positive control, PC] and -100 kcal/kg of the requirement level [negative control, NC]) and three dietary treatments (without additives [CON], CON+CSL [CSL], and CON+CSL+lipase [CSL-Lipase]). Corn and soybean meal-based experimental diets containing vegetable oil were formulated. Growth performance, blood parameters, visceral organ weights, ileal morphology, nutrient digestibility, and cytokine gene expression were measured. RESULTS: Birds fed a diet including CSL increased (p<0.05) lipase level in blood compared to birds fed a diet including CSL-Lipase on day 21. Similarly, higher (p<0.05) liver weight was observed in birds fed a diet including either CSL or CSL-Lipase on day 21. Birds fed NC diet with CSL improved (p<0.05) nutrient digestibility compared to the NC diet on day 21. However, birds fed a diet supplemented with CSL or CSL-Lipase did not affect (p>0.05) the weight gain, feed efficiency, ileal morphology, and cytokine concentrations during the experiment period, regardless of dietary energy levels. CONCLUSION: Our results indicated that CSL has a role in improving nutrient digestibility in young birds when supplemented to a corn-soybean meal based broiler diet.

Responses in growth performance and nutrient digestibility to a multi-protease supplementation in amino acid-deficient broiler diets.

The present study was conducted to investigate the effect of a multi-protease on production indicators of broiler chickens fed a crude protein and amino acid deficient-diets for 35 days immediately after hatch. A total of 448 one-day-old Ross 308 male broiler chicks were allocated in a completely randomized design into one of eight dietary treatments (positive control [PC], negative control [NC: minus 0.5% from PC, and minus 2% of lysine, methionine, threonine and methionine plus cysteine], extreme negative control [ENC: minus 1% from PC, minus 4% of lysine, methionine, threonine and methionine plus cysteine], and plus multi-protease 150 or 300 g per ton [e. g., PC-150]; PC, PC-150, NC, NC-150, NC-300, ENC, ENC-150, ENC-300) to give eight replicates with seven birds in a battery cage. Body weight, average daily gain, average daily feed intake, feed conversion ratio, and mortality were measured every week. Carcass traits, proximate analysis of breast meat, and ileum digestibility were analyzed on day 21 and 35. Feeding a multi-protease (i.e., more than 150 g/ton) for 35 days immediately after hatching improved feed efficiency and ileum digestibility (i.e., dry matter, crude protein, and energy) compared to their counterparts (i.e., diets without multi-protease: PC, NC, and ENC). In conclusion, our results indicated that broiler chickens fed nutrients deficient-diet (i.e., crude protein and amino acids) supplemented a multi-protease had an ability to compensate and (or) improve their growth performance commensurate with increased ileal digestibility for 35 days immediately after hatch.

Physiological impact on layer chickens fed corn distiller's dried grains with solubles naturally contaminated with deoxynivalenol.

OBJECTIVE: An experiment was conducted to investigate the response of laying hens fed corn distiller's dried grains with solubles (DDGS) that are naturally contaminated with deoxynivalenol (DON). METHODS: One hundred and sixty 52-week-old Lohmann Brown Lite hens were randomly allotted to five dietary treatments with 8 replicates per treatment. The dietary treatments were formulated to provide a range of corn DDGS contaminated with DON from 0% to 20% (i.e., 5% scale of increment). All laying hens were subjected to the same management practices in a controlled environment. Body weight, feed intake and egg production were measured biweekly for the entire 8-week experiment. The egg quality was measured biweekly for 8 weeks. On weeks 4 and 8, visceral organ weights, blood metabolites, intestinal morphology, and blood cytokine concentrations were measured. RESULTS: The inclusion of corn DDGS contaminated with DON in the diet did not alter (p> 0.05) the body weight, feed intake, hen-day egg production, egg mass and feed efficiency of the laying hens. No difference was found (p>0.05) in the egg quality of hens that were fed the dietary treatments. Furthermore, hens that were fed a diet containing corn DDGS contaminated with DON showed no change (p>0.05) in the visceral organ weights, the blood metabolites, and the cytokine concentrations. The crypt depth increased (p<0.05) as the amount of corn DDGS contaminated with DON increased. Proportionately, the villus height to crypt depth ratio of the laying hens decreased (p<0.05) with the increasing level of corn DDGS contaminated with DON in the diet. CONCLUSION: The inclusion of corn DDGS contaminated with DON up to 20% in layer diets did not cause changes in egg production performance and egg quality, which indicates that DON is less toxic at the concentration of 1.00 mg DON/kg.

A hot-water extract of Sanguisorba officinalis ameliorates endotoxin-induced septic shock by inhibiting inflammasome activation.

The inflammasome is a multiprotein signaling complex that mediates inflammatory innate immune responses through caspase 1 activation and subsequent IL-1ß secretion. However, because its aberrant activation often leads to inflammatory diseases, targeting the inflammasome holds promise for the treatment of inflammation-related diseases. In this study, it was found that a hot-water extract of Sanguisorba officinalis (HSO) suppresses inflammasome activation triggered by adenosine 5'-triphosphate, nigericin, microbial pathogens, and double stranded DNA in bone marrow-derived macrophages. HSO was found to significantly suppress IL-1ß production in a dose-dependent manner; this effect correlated well with small amounts of caspase 1 and little ASC pyroptosome formation in HSO-treated cells. The anti-inflammatory activity of HSO was further confirmed in a mouse model of endotoxin-induced septic shock. Oral administration of HSO reduced IL-1ß titers in the serum and peritoneal cavity, increasing the survival rate. Taken together, our results suggest that HSO is an inhibits inflammasome activation through nucleotide-binding domain and leucine-rich repeat pyrin domain 3, nucleotide-binding domain and leucine-rich repeat caspase recruitment domain 4 and absent in melanoma 2 pathways, and may be useful for treatment of inflammasome-mediated diseases.

SIRT1-dependent modulation of methylation and acetylation of histone H3 on lysine 9 (H3K9) in the zygotic pronuclei improves porcine embryo development.

BACKGROUND: The histone code is an established epigenetic regulator of early embryonic development in mammals. The lysine residue K9 of histone H3 (H3K9) is a prime target of SIRT1, a member of NAD+-dependent histone deacetylase family of enzymes targeting both histone and non-histone substrates. At present, little is known about SIRT1-modulation of H3K9 in zygotic pronuclei and its association with the success of preimplantation embryo development. Therefore, we evaluated the effect of SIRT1 activity on H3K9 methylation and acetylation in porcine zygotes and the significance of H3K9 modifications for early embryonic development. RESULTS: Our results show that SIRT1 activators resveratrol and BML-278 increased H3K9 methylation and suppressed H3K9 acetylation in both the paternal and maternal pronucleus. Inversely, SIRT1 inhibitors nicotinamide and sirtinol suppressed methylation and increased acetylation of pronuclear H3K9. Evaluation of early embryonic development confirmed positive effect of selective SIRT1 activation on blastocyst formation rate (5.2 ± 2.9% versus 32.9 ± 8.1% in vehicle control and BML-278 group, respectively; P ≤ 0.05). Stimulation of SIRT1 activity coincided with fluorometric signal intensity of ooplasmic ubiquitin ligase MDM2, a known substrate of SIRT1 and known limiting factor of epigenome remodeling. CONCLUSIONS: We conclude that SIRT1 modulates zygotic histone code, obviously through direct deacetylation and via non-histone targets resulting in increased H3K9me3. These changes in zygotes lead to more successful pre-implantation embryonic development and, indeed, the specific SIRT1 activation due to BML-278 is beneficial for in vitro embryo production and blastocyst achievement.

Effects of difructose dianhydride (DFA)-IV on in vitro fertilization in pigs.

Difructose dianhydride IV (DFA-IV) is produced from levan, which is a natural polysaccharide that belongs to the fructan family, through the activity of levan fructotransferase (LF) derived from microorganisms. Recently, DFA-IV has been expected to have diverse applications in the food and medical industry. Here, we examined the potential application of DFA-IV forin vitro fertilization (IVF) in pigs. In the assessment of acrosomal integrity during incubation, intact acrosomal or viable spermatozoa were highly sustained in 0.1% or 0.25% DFA-IV (69.8%-70.8%,P<0.05). Reactive oxygen species (ROS) levels during sperm incubation decreased following the addition of DFA-IV, and 0.1%-0.5% DFA-IV in particular significantly decreased ROS production relative to that seen with no addition or 0.75% DFA-IV. Total fertilization (mono+ polyspermic oocyte) rate was significantly higher in the addition of 0.1% DFA-IV (94.2%) than with other concentrations (71.8%-86.7%,P<0.05). When using reduced IVF times and lower sperm numbers, we found that addition of 0.1%-0.5% DFA-IV significantly increased the fertilization rate (P<0.05). Fertilized oocytes treated with 0.1% DFA-IV exhibited higher embryonic development and blastocyst formation than those treated with other concentrations (P<0.05). Consequently, the addition of DFA-IV during IVF improved fertilization and embryonic development, suggesting the possible use of novel sugars for enhancement of assisted reproductive technology (ART) in mammals.

Evaluation of dietary methionine requirement of male Korean native ducks for 3 weeks post-hatching.

A total of 336 1-day-old male Korean native ducks (KND) were used in a completely randomized design with seven dietary methionine levels (0.30-0.90% with 0.1% increment) to determine the methionine requirement of male Korean native ducks for 3 weeks after hatching. Each dietary treatment had six replicates with eight ducklings per pen. One duckling from each pen (n = 6) was sacrificed to weigh empty body and drumsticks at the end of the experiment. Final body weight and weight gain of 3 weeks old KND were increased with increasing dietary methionine levels up to 0.4%, and then decreased (P < 0.05) with a further increasing dietary methionine level. In contrast, feed conversion ratio of the KND decreased up to 0.4% and increased (P < 0.05) with the increasing dietary methionine level. Both empty body weight and proportions of empty body weight were linearly increased (P < 0.05) while the dietary methionine level elevated up to 0.4%. Estimated dietary methionine requirement for maximum body weights, daily gain and minimum feed conversion ratio were 0.36, 0.39 and 0.40%, respectively, when it was fitted into linear- and quadratic-plateau models.

Potential use of lactic acid bacteria Leuconostoc mesenteroides as a probiotic for the removal of Pb(II) toxicity.

It has been demonstrated that certain lactic acid bacteria (LAB) can sequester metal ions by binding them to their surfaces. In the present study, lead (Pb)-resistant LAB were isolated from kimchi, a Korean fermented food. A total of 96 different LAB strains were isolated, and 52 strains showed lead resistance. Among them, an LAB strain-96 (L-96) identified as Leuconostoc mesenteroides showed remarkable Pb resistance and removal capacity. The maximum adsorption capacity of this strain calculated using the Langmuir isotherm was 60.6 mg Pb/g. In an in vivo experiment, young male mice were provided with water (A), Pb-water (B), or Pb-water+ L-96 (C) during puberty. Lower glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) levels in Pb-exposed male mice that received strain L-96 as a probiotic were suggestive of reduced hepatotoxicity. Moreover, feces from mice treated with L-96 contained more Pb than feces from untreated mice. Increased Pb elimination likely reduced internal accumulation, and this hypothesis was supported by significantly lower Pb concentrations in kidneys and testes of the mice treated with strain L-96. The motility and ATP content of epididymal spermatozoa were partially restored if strain L-96 was administered. In conclusion, isolated L-96 LAB had lead-biosorption activity and efficiently detoxified lead-poisoned male mice, resulting in recovering male reproductive function. These results suggest the potential use of LAB as a probiotic to protect humans from the adverse effects of Pb exposure.

The ART and science of sperm mitophagy.

This article discusses the historical perspective and the new findings of autophagy and ubiquitin-proteasome system cooperation during the post-fertilization sperm mitophagy, a process that eliminates potentially damaged paternal mitochondrial DNA from an early embryo. New insight into the mechanism that promotes clonal, maternal inheritance of mitochondrial genes may be helpful for managing mitochondrial disease and infertility in humans, as well as reproductive performance and production traits in agriculturally important domestic animals.

Autophagy and ubiquitin-proteasome system contribute to sperm mitophagy after mammalian fertilization.

Maternal inheritance of mitochondria and mtDNA is a universal principle in human and animal development, guided by selective ubiquitin-dependent degradation of the sperm-borne mitochondria after fertilization. However, it is not clear how the 26S proteasome, the ubiquitin-dependent protease that is only capable of degrading one protein molecule at a time, can dispose of a whole sperm mitochondrial sheath. We hypothesized that the canonical ubiquitin-like autophagy receptors [sequestosome 1 (SQSTM1), microtubule-associated protein 1 light chain 3 (LC3), gamma-aminobutyric acid receptor-associated protein (GABARAP)] and the nontraditional mitophagy pathways involving ubiquitin-proteasome system and the ubiquitin-binding protein dislocase, valosin-containing protein (VCP), may act in concert during mammalian sperm mitophagy. We found that the SQSTM1, but not GABARAP or LC3, associated with sperm mitochondria after fertilization in pig and rhesus monkey zygotes. Three sperm mitochondrial proteins copurified with the recombinant, ubiquitin-associated domain of SQSTM1. The accumulation of GABARAP-containing protein aggregates was observed in the vicinity of sperm mitochondrial sheaths in the zygotes and increased in the embryos treated with proteasomal inhibitor MG132, in which intact sperm mitochondrial sheaths were observed. Pharmacological inhibition of VCP significantly delayed the process of sperm mitophagy and completely prevented it when combined with microinjection of autophagy-targeting antibodies specific to SQSTM1 and/or GABARAP. Sperm mitophagy in higher mammals thus relies on a combined action of SQSTM1-dependent autophagy and VCP-mediated dislocation and presentation of ubiquitinated sperm mitochondrial proteins to the 26S proteasome, explaining how the whole sperm mitochondria are degraded inside the fertilized mammalian oocytes by a protein recycling system involved in degradation of single protein molecules.

An Antioxidant Davallialactone from Phellinus baumii Enhances Sperm Penetration on In Vitro Fertilization of Pigs.

Davallialactone (DAVA) is a hispidin analogue derived from the medicinal fungus Phellinus baumii. We examined the effect of DAVA on in vitro fertilization (IVF) of pigs. Boar spermatozoa were incubated in fertilization medium with varying concentrations of DAVA, then sperm motility and reactive oxygen species (ROS) level were evaluated. Higher sperm motility was found following the addition of 0.5 or 1 µM DAVA after incubation than addition of other concentrations or controls. ROS level decreased significantly with the addition of DAVA. The rate of normal fertilization was higher in the presence of 1 µM DAVA (65.1%) than were those of other concentrations or controls (45.4~59.4%), and the highest total fertilization rate (mono- and polyspermic oocytes) was observed at 1 µM DAVA (83%). In conclusion, addition of DAVA to fertilization medium improved sperm motility, and reduced ROS level so as to potentially improve sperm-oocyte binding in IVF, suggesting the potential of a compound isolated from mushrooms in assisted reproductive technology for humans and animals.

A hot water extract of Aralia cordata activates bone marrow-derived macrophages via a myeloid differentiation protein 88-dependent pathway and protects mice from bacterial infection.

In traditional Asian medicine, Aralia cordata (AC) is a known as a pain reliever and anti-inflammatory drug. Although several of its biological activities have been reported, the immunomodulatory effects of a hot water extract of AC (HAC) have not yet been described. The aim of this study was to investigate whether HAC modulates the activation of macrophages, which play important roles in innate immune responses against microbial pathogens, and if so, to determine the molecular mechanisms by which HAC mediates this process. It was found that HAC activates bone marrow-derived macrophages (BMDM) and increases amounts of nitric oxide and proinflammatory cytokines in a dose-dependent manner. In addition, HAC was found to induce phosphorylation of NF-κB and mitogen-activated protein kinases (MAPKs), including c-Jun N-terminal kinases, extracellular signal-regulated kinases and p38. Interestingly, these effects were absent in BMDM prepared from myeloid differentiation protein 88-knockout mice. Polysaccharides from HAC exerted stronger immunostimulatory effects than HAC itself. Furthermore, orally administered HAC clearly enhanced clearance of the intracellular pathogen Listeria monocytogenes by boosting innate immune responses. These results demonstrate that HAC exerts immunostimulatory effects through the TLR/MyD88 and NF-κB/MAPK signal transduction pathways.

Phytofabrication of bioinspired zinc oxide nanocrystals for biomedical application.

In the present study, we investigated a novel green route for synthesis of zinc oxide nanoparticles (ZnO NPs) using the extract of young cones of Pinus densiflora as a reducing agent. Standard characterization studies were carried out to confirm the obtained product using UV-Vis spectra, SEM-EDS, FTIR, and XRD. TEM images showed that various shapes of ZnO NPs were synthesized, including hexagonal (wurtzite), triangular, spherical, and oval-shaped particles, with average sizes between 10 and 100 nm. The synthesized ZnO NPs blended with the young pine cone extract have very good activity against bacterial and fungal pathogens, similar to that of commercial ZnO NPs.